Information de reference pour ce titreAccession Number: | 00007529-199801230-00028.
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Author: | Echard, Arnaud; Jollivet, Florence; Martinez, Olivier; Lacapere, Jean-Jacques; Rousselet, Annie; Janoueix-Lerosey, Isabelle; Goud, Bruno
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Institution: | A. Echard, F. Jollivet, O. Martinez, J.-J. Lacapere, A. Rousselet, B. Goud, Unite Mixte de Recherche CNRS 144 et 168, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France. I. Janoueix-Lerosey, INSERM U 248, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France. (Goud) To whom correspondence should be addressed. E-mail: [email protected]. 2 October 1997; accepted 2 December 1997.
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Title: | Interaction of a Golgi-Associated Kinesin-Like Protein with Rab6.[Report]
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Source: | Science. 279(5350):580-585, January 23, 1998.
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Abstract: | Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells.Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)-bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.
Copyright (C) 1998 by the American Association for the Advancement of Science
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12. The BL21(DE3) Escherichia coli strain was transformed with the pET 15b (Novagen, Madison, WI) expression vector containing either his-tagged predicted motor domain (Nt, residues 1 to 530) or his-tagged stalk plus tail domains (Ct, residues 529) to 887) of Rabkinesin-6. Induction was performed for 4 hours at 37[degree sign]C with 0. 3 mM isopropyl-beta-D-thiogalactopyranoside. Purification was achieved by adsorption on nickel beads under native conditions for Nt or denaturing conditions followed by renaturation steps for Ct (QlAexpressionist, QlA-GEN). Purified his-tagged Nt and Ct were then dialzyed against 15 mM imidazole (pH 7), 2 mM MgCl2, 1 mM EGTA, and 1 mM dithiothreitol or 80 mM KOH-Pipes (pH 6. 9), 1 mM MgCl2, and 1 mM EGTA, respectively. Protein concentrations were estimated by the Bradford assay.
13. Tubulin was purified from bovine brain with two cycles of polymerization followed by chromatography on phosphocellulose essentially as described [R. C. Williams and J. C. Lee, Methods Enzymol. 85, 376 (1982)]. Microtubules (1 mg/ml) were polymerized with 20 microM taxol. Microtubule concentration was expressed per tubulin heterodimer. Microtubulebinding assays were performed by incubating for 20 min at 33[degree sign]C purified his-tagged Nt or Ct with taxolstabilized MTs (ratio 1. 5:1 or 1:1, respectively) in binding buffer [80 mM KOH-Pipes (pH 6. 9), 1 mM MgCl2, 1 mM EGTA, 1 mM GTP, and 20 microM taxol]. The samples were then centrifuged in a TLX100 (Beckman, Palo Alto, CA) ultracentrifuge (TLS 55 rotor, 100,000g, 20 min, 33[degree sign]C) on a 15% sucrose cushion. The release of Nt was performed by incubating pellets containing Nt-MT complexes with 10 mM Mg-ATP in the binding buffer for 20 min at 33[degree sign]C, followed by centrifugation under the conditions described above. Equal amounts of pellet and supernatant were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Microtubule-activated ATPase rates were measured at 25[degree sign]C by the reduced form of NAD (+) (NADH) coupled enzymes procedure. His-tagged Nt (55 nM) and increasing concentrations of taxol-stabilized MT (0 to 1. 1 microM) were incubated in a reaction buffer consisting of 20 mM KOH-Pipes (pH 6. 9), 5 mM MgCl2, 1 mM phosphoenolpyruvate, 267 microM beta-NADH, lactate deshydrogenase (0. 1 mg/ml), and pyruvate kinase (0. 1 mg/ml) and supplemented with 1 mM Mg-ATP [the Michaelis constant for ATP in the presence of MTs was found to be 85 microM [9]]. Basal ATPase activity was determined at 25[degree sign]C in the same reaction buffer with increasing concentrations of his-tagged Nt (from 55 to 930 nM).
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15. A polyclonal rabbit antiserum was raised to purified glutathione S-transferase-tagged 174 protein expressed in E. coli. HeLa cells were mechanically broken with a barrel-type homogenizer. Total extract, postnuclear supernatant (PNS), high speed pellet, and supernatant were prepared in 50 mM Hepes (pH 7.1) and 90 mM KCl with protease inhibitors, resolved by SDS-PAGE, and immunoblotted essentially as described [M. Roa, V. Cornet, C. Z. Yang, B. Goud, J. Cell Sci. 106, 789 (1993)].
16. HeLa cells were fixed in methanol for 4 min at -20[degree sign]C and processed for confocal laser scanning microscopy and immunofluorescence analysis as described [6]. Images were recorded and imported into Adobe Photoshop 4. 0 for compilation.
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18. HeLa cells were transfected with pEGFP-Rabkinesin-6 for 18 hours, with the DOTAP reagent (Boehringer Mannheim). To obtain Rabkinesin-6 fused at its NH2 -terminus with GFP, we introduced the blunted SMa I-Hind III fragment from pGEM-Rabkinesin-6 into the Sma 1-digested pEGFP-C1 plasmid (Clontech). pGEM-Rabkinesin-6 was obtained after the creation of a Sma I site upstream of the initiator methionine of Rabkinesin-6 cDNA by polymerase chain reaction amplification with the primers 5'-GAACCCGGGAATGTCTCACCGGATCCTT-3' and 5'-GGGAATTCGAAGGTAAACTTTC-3', followed by cloning into Sma I-Eco RI sites of the pGEM-4Z plasmid (Promega).
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20. HeLa cells were transfected with pGEM-Nt or pGEM-Ct for 4 hours, with the vaccinia system [6]. To obtain pGEM-Nt and pGEM-Ct, we first added the double-strand DNA linker 5'-CCCGGGAGCCATGGTTCCTCAGGTTTGAGGTACCGAATTC-3' between the Sma I and Eco RI sites of the pGEM-4Z plasmid to create a Bsu 36I restriction site, a stop codon, and an initiation codon in an optimal Kozak's context (pGEM4Z*). pGEM-Nt was obtained by introducing the Sma I-Bsu 36I fragment from pGEM-Rabkinesin-6 into pGEM4Z*, pGEM-Ct was obtained by introducing the Bsu 36I digestion fragment of pGEM-Rabkinesin-6 into Bsu 36I-digested pGEM4Z*. pGEM-myc-Nt and pGEM-myc-Ct were constructed by ligating the double-strand DNA linker 5'-GCATGCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGAATGACCCGGG-3' (encoding for MEQKLISEEDLN) [7] into Sph I-Sma I-digested pGEM-Nt and pGEM-Ct plasmids, respectively.
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22. pGEM-myc-Rabkinesin-6 was constructed by ligating the double-strand DNA linker 5'-GCATGCCACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGAATGACCCGGG-3' (encoding for MEQKLISEEDLN) [7] into Sph I-Sma I-digested pGEM-Rabkinesin-6 plasmid. HeLa cells were cotransfected with the vaccinia system [6] with pGEM-myc-Rabkinesin-6, pGEM-myc-Nt [20], or pGEM-myc-Ct [20] plasmids (1.5 microg/well) and either pGEM-1 (control) or plasmids encoding for Rab6 constructs [6] (1.5 microg/well). After 4 hours, cells were incubated for 15 min in medium without methionine and cysteine (ICN Pharmaceuticals, Costa Mesa, CA) and metabolically labeled for 20 min with [(35) S]methionine (250 microCi/ml) and [(35) S]cysteine (250 microCi/ml) (Amersham). After a 30-min chase in complete medium supplemented with 2. 5 mM methionine and cysteine, cells were lysed in 1% Nonidet P-40 (Sigma), 20 mM Hepes (pH 7. 4), 150 mM NaCl, 5 mM MgCl2, 0. 2% bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors by incubation at 4[degree sign]C for 10 min. Supernatants obtained after centrifugation for 10 min at 20,000g were precleared by incubation with protein-G Sepharose for 30 min at 4[degree sign]C. After preclearing, the supernatants were incubated with 9E10 anti-myc (and in one experiment with affinity-purified anti-Rab6) together with protein-G Sepharose beads for 1 hour at 4[degree sign]C. The immunoprecipitates were then washed 4 times in the lysis buffer and analyzed by SDS-PAGE and autoradiography.
23. Transport of SEAP was monitored essentially as described [6]. Briefly, HeLa cells cotransfected with pGEM-SEAP and various constructs encoding plasmids were labeled for 4 hours with [(35) S]methionine (50 microCi/ml) and [(35) S]cysteine (50 microCi/ml). SEAP present in cell lysates or in the medium was immunoprecipitated with a polyclonal antibody to calf intestine alkaline phosphatase (Rockland). Immunoprecipitates were then digested with endoglycosidase H (endo H) (Sigma). Endo H treatment allowed us to distinguish two intracellular forms of SEAP: SEAP molecules bearing endo H-sensitive oligosaccharides still present in compartments before cis-medial Golgi (mainly ER) and endo H-resistant molecules (also sensitive to neuraminidase) corresponding to SEAP that have reached late Golgi-TGN compartments, Immunoprecipitates were then analyzed by SDS-PAGE and autoradiography. Bands corresponding to SEAP were quantified with a Phosphorimager (Molecular Dynamics) equipped with the Image Quant software.
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25. The conserved domains are RXRP (amino acids 69 to 72), YGQ(T/S)X(T/S/A)GK(T/S) (amino acids 158 to 166, except for the Q replaced by a V as in CHO1 or HMKLP1), NXXSSRSH (amino acids 373 to 380), and DLAGXE (amino acids 407 to 412) [7]. Highly conserved residues EXYXE/DXXXDLL (amino acids 306 to 316) and (I/V)P(F/Y)R (amino acids 456 to 459), possibly corresponding to MT-binding sites, are also present [E. P. Sablin, F. J. Kull, R. Cooke, R. D. Vale, R. J. Fletterick, Nature 380, 555 (1996)].
26. A. Lupas, M. Van Dyke, J. Stock, Science 252, 1162 (1991).
27. We thank M. Bornens, M.-H. Cuif, B. Hoflack, L. Johannes, G. Langsley, J. de Mey, M. Kress, J.-L. Rigaud, J. Salamero, and C. Saudrais for critical reading of this manuscript and helpful discussions; M. Bornens (Institut Curie, Paris), E. Berger (Zurich, Switzerland), M. Zerial [European Molecular Biology Laboratory (EMBL), Heidelberg, Germany], and P. Chavrier (Centre d'Immunologie de Marseille-Luminy, Marseille, France) for the gifts of CTR 433 monoclonal antibody, affinity-purified polyclonal antibody to human beta-1,4-galactosyltransferase, pLexA-Rab5 Q79L, and pLexA-Rab7 Q67L, respectively; and G. Bordenave for help in rabbit immunization. Supported in part by grants from the Association de la Recherche Contre le Cancer, the Fondation de la Recherche Medicale, the European Community, and the Human Frontier Science Program.
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Language: | English.
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Document Type: | Reports.
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Journal Subset: | Life Sciences. Physical Science & Engineering.
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ISSN: | 0036-8075
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NLM Journal Code: | 0404511, uj7
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